Ubiquitin-dependent proteolysis regulates diverse biological processes such as the cell cycle, signaling pathways, tumor suppressor and oncogene networks and the immune response. The ubiquitination of a target protein proceeds through a multi-enzyme pathway involving ubiquitin-activating (El), ubiquitin-conjugating (E2) and ubiquitin-protein ligase (E3) activities. The E3 is responsible for target specificity, and represents the primary regulated step in the process. E3s comprise a large superfamily of proteins and protein complexes. RING E3s are the biggest and most diverse class of E3s. They promote ubiquitination in a reaction that is poorly understood. This proposal focuses on the crystallographic and biochemical characterization of four families of RING E3s, of their complexes with substrates and E2s, and of ubiquitination reaction intermediates. These are the 4-protein SGF, the 5-protein VHL/SOCS, the 10-protein APC and the single chain c-CbI proto-oncogene. The SCF E3 consists of an F-box protein, Skp1, Cull and Rbxl, and is by far the largest E3 family known to date, with hundreds of SCF subunits in the databases. Structures of two human SCFs, SCFSKp2 and SCFbTRCP, which ubiquitinate the p27Kipl tumor suppressor and the beta-catenin oncoprotein, respectively, will be determined. A divergent SCF-like E3 is exemplified by the VHL tumor suppressor, which binds ElonginC, ElonginB, Cul2 and Rbx1, and induces the degradation of the hypoxia-inducible transcription factor 1alpha (HIF1), negatively regulating angiogenesis. The structure of the entire VHL complex bound to an HIF1 substrate peptide will be determined. The APC (anaphase promoting complex) E3 has a central role in coordinating mitosis. Two APC subunits, Apc2 and Apc1 1, share limited homology to the Cul1-Rbx1 components of the SCF. The function of the other subunits is not yet known. In the first step, the structure of the Apc2-Apc1 1 catalytic core will be determined. Several additional subunits that interact with the core will then be assembled and their structures determined. Cbl ubiquitinates the activated forms of several receptor tyrosine kinases (RTKs) and terminates signaling. The minimal portion of PDGFR that binds to CbI and gets ubiquitinated will be determined. Building on the Cbl-E2 structure, the structure of the CbI-E2-substrate complex will be determined. These studies will address questions about substrate recognition, mechanism(s) of ubiquitination, specificity in ubiquitination sites and between different E3 subunits, and the architectures of E3s, with implications for the function and regulation of these large and diverse complexes. They will also help address whether E3s, many of which are implicated in diseases such as cancer, are likely targets for drug discovery.